Patterned binding of functionalized microspheres for optical diffraction-based biosensors

ABSTRACT

The present invention provides an inexpensive and sensitive system and method for detecting analytes present in a medium. The system comprises a diffraction enhancing element, such as functionalized microspheres, which are modified such that they are capable of binding with a target analyte. Additionally, the system comprises a polymer film, which may include a metal coating, upon which is printed a specific, predetermined pattern of a analyte-specific receptors. Upon attachment of a target analyte to select areas of the polymer film, either directly or with the diffraction enhancing element, diffraction of transmitted and/or reflected light occurs via the physical dimensions and defined, precise placement of the analyte. A diffraction image is produced which can be easily seen with the eye or, optionally, with a sensing device.

TECHNICAL FIELD

The present invention is generally in the field of detecting analytes ina medium and, more particularly, the present invention relates to theuse of functionalized microspheres for enhancing optical diffractionwith single use, disposable sensors to indicate the presence of theanalyte in a medium.

BACKGROUND OF THE INVENTION

There are many systems and devices available for detecting a widevariety of analytes in various media. Most of these systems and devicesare relatively expensive and require a trained technician to perform thetest. There are many cases where it would be advantageous to be able torapidly and inexpensively determine if an analyte were present. What isneeded is a biosensor system that is easy and inexpensive to manufactureand is capable of reliable and sensitive detection of analytes,including smaller analytes.

Sandstrom et al., 24 Applied Optics 472, 1985, describe use of anoptical substrate of silicon with a layer of silicon monoxide and alayer of silicon formed as dielectric films. They indicate that a changein film thickness changes the properties of the optical substrate toproduce different colors related to the thickness of the film. Thethickness of the film is related to the color observed and a filmprovided on top of an optical substrate may produce a visible colorchange. The authors indicate that a mathematical model can be used toquantitate the color change, and that “[c]alculations performed usingthe computer model show that very little can be gained in opticalperformance from using a multilayer structure . . . but a biolayer onthe surface changes the reflection of such structures very little sincethe optical properties are determined mainly by the interfaces insidethe multilayer structure. The most sensitive system for detection ofbiolayers is a single layer coating, while in most other applicationsperformance can be by additional dielectric layers.”

Sandstrom et al., go on to indicate that slides formed from metal oxideson metal have certain drawbacks, and that the presence of metal ions canalso be harmful in many biochemical applications. They indicate that theideal top dielectric film is a 2-3 nm thickness of silicon dioxide whichis formed spontaneously when silicon monoxide layer is deposited inambient atmosphere, and that a 70-95 nm layer silicon dioxide on a 40-60nm layer of silicon monoxide can be used on a glass or plasticsubstrate. They also describe formation of a wedge of silicon monoxideby selective etching of the silicon monoxide, treatment of the silicondioxide surface with dichlorodimethylsilane, and application of abiolayer of antigen and antibody. From this wedge construction they wereable to determine film thickness with an ellipsometer, and note that the“maximum contrast was found in the region about 65 nm where theinterference color changed from purple to blue.” They indicate that thesensitivity of such a system is high enough for the detection of proteinantigen by immobilized antibodies. They conclude “the designs given aresensitive enough for a wide range of applications. The materials, i.e.,glass, silicon, and silicon oxides, are chemically inert and do notaffect the biochemical reaction studied. Using the computations above itis possible to design slides that are optimized for differentapplications. The slides can be manufactured and their quality ensuredby industrial methods, and two designs are now commercially available.

U.S. Pat. No. 5,512,131 issued to Kumar et al. describes a device thatincludes a polymer substrate having a metal coating. An analyte-specificreceptor layer is stamped on the coated substrate. The device is used ina process for stamping or as a switch. A diffraction pattern isgenerated when an analyte binds to the device. A visualization device,such as a spectrometer, is then used to determine the presence of thediffraction pattern.

However, the device described by Kumar et al. has several disadvantages.One disadvantage is that an extra visualization device is needed to viewany diffraction pattern. By requiring a visualization device, the Kumaret al. device does not allow a large number of samples to be testedsince it is not possible to determine the presence of an analyte byusing the unaided eye. Additionally, this device is not able to detectsmaller analytes as these analytes do not produce a noticeablediffraction pattern.

U.S. Pat. No. 5,482,830 to Bogart, et al., describes a device thatincludes a substrate which has an optically active surface exhibiting afirst color in response to light impinging thereon. This first color isdefined as a spectral distribution of the emanating light. The substratealso exhibits a second color which is different from the first color (byhaving a combination of wavelengths of light which differ from thatcombination present in the first color, or having a different spectraldistribution, or by having an intensity of one or more of thosewavelengths different from those present in the first color). The secondcolor is exhibited in response to the same light when the analyte ispresent on the surface. The change from one color to another can bemeasured either by use of an instrument, or by eye. Such sensitivedetection is an advance over the devices described by Sandstrom andNygren, supra, and allow use of the devices in commercially viable andcompetitive manner.

However, the method and device described in the Bogart, et al. patenthas several disadvantages. One disadvantage is the high cost of thedevice. Another problem with the device is the difficulty in controllingthe various layers that are placed on the wafer so that one obtains areliable reading.

Additionally, biosensors having a self-assembling monolayer have beenused to detect analytes and are set forth in U.S. patent applicationSer. Nos. 08/768,449 and 08/991,844, both of which are incorporatedherein by reference in their entirety. However, these biosensorscurrently do not have the requisite sensitivity required to detectsmaller analytes since these smaller analytes do not produce asufficient diffraction pattern to be visible.

Some commercial lateral flow technologies have been used which employlatex bead technology. These technologies are currently employed in mostof the commercially-available home diagnostic kits (e.g. pregnancy andovulation kits). These kits use colored beads which accumulate in adefined “capture zone” until the amount of beads becomes visible to theunaided eye. However, these systems lack the requisite sensitivity totest for many analytes, since a much larger number of latex beads mustbind in the capture zone to be visible to the naked eye than thatrequired to cause diffraction in the same size zone. Generally, thenumber of beads needed is about 2 to 3 orders of magnitude higher thanthe sensors of the present invention.

What is needed is a biosensor system that is easy and inexpensive tomanufacture and is capable of reliable and sensitive detection ofanalytes, including smaller analytes.

SUMMARY OF THE INVENTION

The present invention provides an inexpensive and sensitive system andmethod for detecting analytes present in a medium. The system comprisesa biosensing device having a polymer film upon which is printed aspecific, predetermined pattern of analyte-specific receptors. Thepolymer film may be coated with a metal layer. Additionally, the systemutilizes “diffraction enhancing elements” which are capable of bindingto the target analyte and to the biosensor and are capable of producinga substantial change in the height and/or refractive index, therebyincreasing the diffraction efficiency of the biosensor and permittingthe detection of smaller analytes. In use, a target analyte attacheseither to the diffraction enhancing element, which then attaches to thebiosensor, or directly to select areas of the polymer film upon whichthe receptor is printed. Then diffraction of transmitted and/orreflected light occurs via the physical dimensions and defined, preciseplacement of the analyte. A diffraction image is produced which can beeasily seen with the eye or, optionally, with a sensing device.

The system of the present invention is much more sensitive than currentinexpensive systems. The system of the present invention is able todetect low to high molecular weight analytes, microorganisms, and DNA orRNA species in fluid samples. More specifically, the system is able todetect hormones, steroids, antibodies, drug metabolites, and evennucleic acids, among others. This is a significant expansion of theoptical diffraction-based sensing technology set forth in U.S. patentapplication Ser. Nos. 08/768,449 and 08/991,844.

The present invention utilizes diffraction enhancing elements, such aslatex microspheres, which aid in the detection of smaller analytes.Normally, after an analyte binds to an analyte-specific receptor on abiosensor, the analyte will diffract or reflect transmitted light toproduce a diffraction pattern. If the analyte is larger, the diffractionpattern is able to be seen with the unaided eye. However, some analytesare too small such that the diffraction pattern produced is not able tobe seen. By using diffraction enhancing elements, the biosensor havingthe analyte-specific receptor material may be used to detect thesesmaller analytes. The diffraction enhancing elements used are capable ofbinding to the analyte, and then the element with bound analyte binds tothe biosensor. Then, as the light is transmitted through or reflectedfrom the biosensor, the element enhances the diffraction patterngenerated by the analyte such that the resulting diffraction pattern maybe seen by the unaided eye.

The present invention also utilizes methods of contact printing ofpatterned, analyte-specific receptors. The analyte-specific receptorshave receptive materials bound thereto. The receptive materials arespecific for a particular analyte or class of analyte, depending uponthe receptor used. Methods of contact printing which would be useful ingenerating the sensing devices used in the present system are disclosedfully in U.S. patent application Ser. Nos. 08/707,456 and 08/769,594,both of which are incorporated herein by reference in their entirety.However, since these methods relate to self-assembling monolayers, themethods need to be altered slightly, as discussed below, to print theanalyte-specific receptor material as this material is notself-assembling.

Patterned analyte-specific receptor layers allow for the controlledplacement of analytes and/or diffraction enhancing elements thereon viathe patterns of analyte-specific receptors. The biosensing devices ofthe present invention produced thereby are used by first exposing thebiosensing device to a medium that contains the analyte of choice mixedwith the diffraction enhancing element. Then, after an appropriateincubation period, a light, such as a laser or other point light source,is transmitted through or reflected from the film. If the analyte ispresent in the medium and is bound, either directly or in conjunctionwith the diffraction enhancing element, to the receptors on thepatterned analyte-specific receptor layer, the light is diffracted insuch a way as to produce a visible image. In other words, theanalyte-specific receptor layers with the analyte and/or diffractionenhancing element bound thereto can produce optical diffraction patternswhich differ depending on the reaction of the receptors on theanalyte-specific receptor layer with the analyte of interest. The lightcan be in the visible spectrum, and be either reflected from the film,or transmitted through it, and the analyte can be any compound orparticle reacting with the analyte-specific receptor layer. The lightcan be a white light or monochromatic electromagnetic radiation in thevisible region. While visible light is the desired light source, thepresent invention may also be used with non-visible point light sources,such as near-infrared light, coupled with a detector. The thickness ofthe film and the size of the microparticle may be adjusted to compensatefor the non-visible light source. Additionally, the present inventionalso provides a flexible support for an analyte-specific receptor layereither directly on the substrate or on gold or other suitable metal ormetal alloy.

The present invention provides an analyte-specific receptor layer ongold or other material which is suitable for mass production. Thebiosensors used in the present invention can be produced as a singletest for detecting an analyte or it can be formatted as a multiple testdevice. The biosensors of the present invention can be used to detect(1) antigens or antibodies associated with medical conditions, (2)contamination in garments, such as diapers, and (3) contamination bymicroorganisms.

In another embodiment of the present invention, nutrients for a specificclass of microorganisms can be incorporated into the analyte-specificreceptor layer. In this way, very low concentrations of microorganismscan be detected by first contacting the biosensor of the presentinvention with the nutrients incorporated therein and then incubating,if necessary, the biosensor under conditions appropriate for the growthof the bound microorganism. The microorganism is allowed to grow untilthere are enough organisms to form a diffraction pattern.

The present invention can also be used on contact lenses, eyeglasses,window panes, pharmaceutical vials, solvent containers, water bottles,adhesive bandages, and the like to detect contamination.

These and other features and advantages of the present invention willbecome apparent after a review of the following detailed description ofthe disclosed embodiments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a biosensor capable of simultaneously measuring severaldifferent analytes in a medium.

FIG. 2 is a schematic of contact printing of analyte-specific receptorlayers.

FIG. 3 is an atomic force microscopy image of evaporated gold on MYLAR®,purchased from Courtaulds Performance Films (Canoga Park, Calif.). Theaverage roughness of the gold layer is 3-4 nanometers, with maximumroughness of 9 nanometers.

FIG. 4 is an SEM photomicrograph showing patterned attachment ofdiffraction enhancing elements in the presence of an analyte.

DETAILED DESCRIPTION

The present invention features improved biosensing devices, and methodsfor using such biosensing devices, for detecting and quantifying thepresence or amount of an analyte of interest within a medium. Thepresent invention is much more sensitive and can be used to detectsmaller analytes which, until now, were not able to be detected withoutthe use of expensive instruments. The analytes that can be detected bythe present invention include, but are not limited to, hormones,proteins such as antibodies, steroids, drug metabolites, nucleic acids,microorganisms such as bacteria, yeasts, fungi and viruses. In contrastto prior devices, those of the present invention allow detection ofextremely small quantities and sizes of analytes in a medium in a rapidassay lasting only a few minutes. In addition, no signaling orassociated electronic components are required in the present invention.

The present invention comprises micro-contact printing ofanalyte-specific receptors onto polymer film, which may have a metalcoating thereon. The invention allows for the development of single use,disposable biosensors based on light diffraction to indicate thepresence of the analyte. Additionally, the present invention includesdiffraction enhancing elements which increase the diffraction efficiencyof the biosensor, thereby making it possible to detect any number ofdifferent analytes. Upon attachment of a target analyte to select areasof the polymer film which contain the receptor, either directly or incombination with a diffraction enhancing element, diffraction oftransmitted and/or reflected light occurs via the physical dimensionsand defined, precise placement of the analyte. For example, yeast, fungior bacterium are large enough to act as diffraction elements for visiblelight when placed in organized patterns on a surface. However, smalleranalytes, such as viruses, proteins, molecules, hormones, steroids, drugmetabolites and nucleic acids, are only capable of acting as suitablediffraction elements when they are also bound to a diffraction enhancingelement. In addition to producing a simple diffraction image, patternsof analytes can be such as to allow for the development of a holographicsensing image and/or a change in visible color. Thus, the appearance ofa hologram or a change in an existing hologram will indicate a positiveresponse. The pattern made by the diffraction of the transmitted lightcan be any shape including, but not limited to, the transformation of apattern from one pattern to another upon binding of the analyte to thereceptive material. In particularly preferred embodiments, thediffraction pattern is discernible in less than one hour after contactof the analyte with the biosensing device of the present invention.

The diffraction grating which produces the diffraction of light uponinteraction with the analyte and/or element should have a minimumperiodicity of the wavelength of incident light. Very small analytes canbe detected indirectly by using diffraction enhancing element particlesthat are specific for the small analyte. One embodiment in which thesmall analyte can be detected comprises coating the element particle,such as a latex bead, with a receptor material that specifically bindsto the analyte of interest.

A variety of methods may be used to attach the receptor material ontothe diffraction enhancing particle. These methods include, but are notlimited to, simple physisorption to a hydrophobic particle (e.g.,binding a protein onto polystyrene particles); binding using a protein Aor protein G linker; binding using a streptavidin or avidin-biotinlinker; or binding using covalent attachment. A preferred embodiment ofthe present invention is to use carbodiimide coupling of a proteinaceousreceptor to carboxylated particles. Other methods of coupling well-knownto those of ordinary skill in the art may be used as well.

Diffraction enhancing element particles that can be used in the presentinvention include, but are not limited to, glass, cellulose, syntheticpolymers or plastics, latex, polystyrene, polycarbonate, bacterial orfungal cells and the like. The particles are preferably spherical inshape, but the structural and spatial configuration of the particle isnot critical to the present invention. For instance, the particles couldbe slivers, ellipsoids, cubes, and the like. A desirable particle sizeranges from a diameter of approximately 0.1 μm to 100.0 μm, desirablybetween approximately 0.3 μm to 1 μm. The composition of the elementparticle is not critical to the present invention. Preferably, thedifference in refractive index between the medium and the enhancingelement is between 0.1 and 1.0. More preferably, the difference inrefractive index between the medium and the enhancing element is between0.2 and 0.7.

The analyte-specific receptor layer on the polymer film contains areceptive material, such as an antibody, that will specifically bind toan epitope on the analyte that is different from the epitope used in thebinding to the particle. Thus, for detecting a small analyte, such asviral particles, the medium is first exposed to the diffractionenhancing element particles, such as latex particles, to which the viralparticles bind. Then, the diffraction enhancing element particles areoptionally washed and exposed to the polymer film with theanalyte-specific receptor layers containing the virus specificantibodies. The antibodies then bind to the viral particles on theelement particle thereby immobilizing the element particles in the samepattern as the receptors on the film. Because the bound elementparticles will cause diffraction of the visible light, a diffractionpattern is formed, indicating the presence of the viral particle in theliquid. Additionally, the polymer film may include a metal coatingthereon. The analyte-specific receptor layer would then be located onthe metalized surface of the film.

Alternatively, the analyte may be detected by first exposing thesubstrate to the medium containing the analyte and causing the analyteto bind to the analyte-specific receptor layer material. Next, asolution containing the diffraction enhancing element particles iscontacted with the substrate having the analyte bound thereto. Theparticles then bind to the analyte. Because the bound element particleswill cause diffraction of the visible light, a diffraction pattern isformed, indicating the presence of the analyte in the liquid.

Finally, in a preferred embodiment, the biosensor, the diffractionenhancing element particles and the medium containing the analyte may beadmixed simultaneously. This will result in a combination of the bindingprocedures discussed above. Some of the analytes will first bind with adiffraction enhancing element particle prior to binding to thesubstrate. Other analytes will first bind with the substrate and thenbind with an element particle. When a point-light source is shownthrough the sensor, a diffraction pattern is formed, indicating thepresence of the analyte in the liquid.

The analytes that are contemplated as being detected using the presentinvention include, but are not limited to, bacteria; yeasts; fungi;viruses; rheumatoid factor; antibodies, including, but not limited toIgG, IgM, IgA and IgE antibodies; carcinoembryonic antigen;streptococcus Group A antigen; viral antigens; antigens associated withautoimmune disease; allergens; tumor antigens; streptococcus Group Bantigen; HIV I or HIV II antigen; or host response (antibodies) to theseand other viruses; antigens specific to RSV or host response(antibodies) to the virus; an antigen; enzyme; hormone; polysaccharide;protein; lipid; carbohydrate; drug or nucleic acid; Salmonella species;Candida species, including, but not limited to Candida albicans andCandida tropicalis; Salmonella species; Neisseria meningitides groups A,B, C, Y and W sub 135, Streptococcus pneumoniae, E. coli K1, Haemophilusinfluenza type B; an antigen derived from microorganisms; a hapten, adrug of abuse; a therapeutic drug; an environmental agent; and antigensspecific to Hepatitis.

In another embodiment of the present invention, nutrients for a specificclass of microorganisms can be incorporated into the analyte-specificreceptor layer. In this way, very low concentrations of microorganismscan be detected by first contacting the biosensor of the presentinvention with the nutrients incorporated therein and then incubatingthe biosensor under conditions appropriate for the growth of the boundmicroorganism. The microorganism is allowed to grow until there areenough organisms to form a diffraction pattern. Of course, in somecases, the microorganism is present or can multiply enough to form adiffraction pattern without the presence of a nutrient on the patternedmonolayer.

A part of the present invention is the analyte-specific receptormaterial that can be microprinted on the polymer film and willspecifically bind to the analyte of interest. Thus, the receptormaterial is defined as one part of a specific binding pair and includes,but is not limited to, antigen/antibody, enzyme/substrate,oligonucleotide/DNA, chelator/metal, enzyme/inhibitor,bacteria/receptor, virus/receptor, hormone/receptor, DNA/RNA, orRNA/RNA, oligonucleotide/RNA, and binding of these species to any otherspecies, as well as the interaction of these species with inorganicspecies. Additionally, when a metalized polymer film is used, theanalyte-specific receptor material can be microprinted on the metalizedsurface of the film.

The receptor material that is bound to the attachment layer ischaracterized by an ability to specifically bind the analyte or analytesof interest. The variety of materials that can be used as receptormaterial are limited only by the types of material which will combineselectively (with respect to any chosen sample) with the analyte.Subclasses of materials which can be included in the overall class ofreceptor materials includes toxins, antibodies, antigens, hormonereceptors, parasites, cells, haptens, metabolites, allergens, nucleicacids, nuclear materials, autoantibodies, blood proteins, cellulardebris, enzymes, tissue proteins, enzyme substrates, coenzymes, neurontransmitters, viruses, viral particles, microorganisms, proteins,polysaccharides, chelators, drugs, and any other member of a specificbinding pair. This list only incorporates some of the many differentmaterials that can be coated onto the attachment layer to produce a thinfilm assay system. Whatever the selected analyte of interest is, thereceptor material is designed to bind with the analyte of interest. Inthe preferred embodiments, the biosensing device is configured andarranged to provide a pattern detectable by eye in response totransmission of a point light source when the analyte of interest issandwiched between the receptor material and a diffraction enhancingelement.

In many instances, a “blocker” may be necessary to prevent non-specificbinding. The term “blocker” as used herein means a reagent that adheresto the sensor surface so that it “blocks” or prevents non-analytematerials from binding to the surface (either in the patterned orun-patterned areas). The blocking step may be done as a post-treatmentto a surface which has already been contact printed (“post-block”), andis the standard technique for filling in non-contact printed regionswith another thiol. However, the inventors have discovered that a“pre-block” technique is preferred over the post-block technique. In thepre-block technique, the surface of the substrate is pre-treated with anon-thiol containing blocker and then contact printed. Not wishing to bebound to any theory, it is theorized that the contact printed material(usually sulfur containing) displaces the physisorbed blocker, therebypermitting the analyte-specific receptor material to be bound directlyto the surface of the substrate. A subsequent post-block may also beperformed, if desired. Blockers can include, but are not limited to,β-casein, albumins such as bovine serum albumin, pluronic or othersurfactants, polyethylene glycol, polyvinyl alcohol, or sulfurderivatives of the above compounds, and any other blocking materialknown to those of ordinary skill in the art.

The matrix containing the analyte of interest may be an interstitialfluid, a solid, a gas, or a bodily fluid such as mucous, saliva, urine,fecal material, tissue, marrow, cerebral spinal fluid, serum, plasma,whole blood, sputum, buffered solutions, extracted solutions, semen,vaginal secretions, pericardial, gastric, peritoneal, pleural, a throatswab or other washes and the like. The analyte of interest may be anantigen, an antibody, an enzyme, a DNA fragment, an intact gene, a RNAfragment, a small molecule, a metal, a toxin, an environmental agent, anucleic acid, a cytoplasm component, pili or flagella component,protein, polysaccharide, drug, or any other material. For example,receptor material for bacteria may specifically bind a surface membranecomponent, protein or lipid, a polysaccharide, a nucleic acid, or anenzyme. The analyte which is indicative of the bacteria may be asaccharide or polysaccharide, an enzyme, a nucleic acid, a membranecomponent, a ganglioside or an antibody produced by the host in responseto the bacteria. The presence of the analyte may indicate an infectiousdisease (bacterial or viral), cancer, an allergy, or other medicaldisorder or condition. The presence of the analyte may be an indicationof water or food contamination or other harmful materials. The analytemay indicate drug abuse or may monitor levels of therapeutic agents.

One of the most commonly encountered assay protocols for which thistechnology can be utilized is an immunoassay. However, the generalconsiderations apply to nucleic acid probes, enzyme/substrate, and otherligand/receptor assay formats. For immunoassays, an antibody may serveas the receptor material and/or it may be the analyte of interest. Thereceptor material, for example an antibody or an antigen, must form astable, reactive layer on the attachment layer of the test device. If anantibody is the receptor material, the antibody must be specific to theantigen of interest; and the antibody (receptor material) must bind theantigen (analyte) with sufficient avidity that the antigen is retainedat the test surface. In some cases, the analyte may not simply bind thereceptor material, but may cause a detectable modification of thereceptor material to occur. This interaction could cause an increase inmass at the test surface or a decrease in the amount of receptormaterial on the test surface. An example of the latter is theinteraction of a degradative enzyme or material with a specific,immobilized substrate. In this case, one would see a diffraction patternbefore interaction with the analyte of interest, but the diffractionpattern would disappear if the analyte were present. The specificmechanism through which binding, hybridization, or interaction of theanalyte with the receptor material occurs is not important to thisinvention, but may impact the reaction conditions used in the finalassay protocol.

In general, the receptor material may be passively applied to thesubstrate layer. If required, the free functional groups introduced ontothe test surface by the attachment layer may be used for covalentattachment of receptor material to the test surface.

A wide range of techniques can be used to apply the receptor material tothe substrate layer. Test surfaces may be coated with receptor materialby application of solution in discrete arrays or patterns; spraying, inkjet, contact printing or other imprinting methods; or printing a blockermaterial in a pattern followed by total immersion or spin coating withthe receptor material. The technique selected should minimize the amountof receptor material required for coating a large number of testsurfaces and maintain the stability/functionality of receptor materialduring application. The technique must also apply or adhere the receptormaterial to the attachment layer in a very uniform and controlledfashion.

The biosensing device of the present invention utilizes methods ofcontact printing of patterned, analyte-specific receptor layers onpolymer or metalized polymer films, desirably transparent orsemi-transparent, the compositions produced thereby, and the use ofthese compositions. Patterned analyte-specific receptor layers allow forthe controlled attachment (or binding) placement of the analytereceptor. The term “patterned analyte-specific receptor layers thereon”as used herein means the analyte-specific receptor layers in any patternon the polymer or metalized polymer films, including a solid pattern.

When the film with the patterned analyte-specific receptor layersthereon is exposed to an analyte that is capable of reacting with theanalyte-specific receptor layer, the film will produce opticaldiffraction patterns which differ depending on the reaction of thepatterned analyte-specific receptor layer with the analyte of interest.The medium would contain the diffraction enhancing element particles.The medium may be a high surface tension fluid such as water. The lightcan be in the visible spectrum, and be either reflected from the film,or transmitted through it, and the analyte can be any compound reactingwith the analyte-specific receptor layer.

In preferred embodiments, the method involves contacting the sensingdevice with a test sample containing the diffraction enhancing elementparticles and potentially containing the analyte. If the analyte ispresent in the sample, then when light is transmitted through ametalized polymer film with the analyte-specific receptor layer, avisible diffraction image is formed.

The medium in which the analyte may reside can be solid, gel-like,liquid or gas. For purposes of detecting an analyte in a body fluid, thefluid is selected from, but not limited to, urine, serum, plasma, spinalfluid, sputum, whole blood, saliva, uro-genital secretions, fecalextracts, pericardial, gastric, peritoneal, pleural washes, vaginalsecretions, or a throat swab. The most common gas that is contemplatedas being used with the biosensing device of the present invention isair.

In one embodiment, the present invention is contemplated in a dipstickform in which a micro-contact printed metalized film is mounted at theend of the dipstick. In use, the dipstick is dipped into the liquid inwhich the suspected analyte may be present. The liquid would alsocontain the diffraction enhancing element particles. The dipstick isallowed to remain for several minutes. The dipstick is then removed andthen, either a light is projected through the metalized film or the filmis observed with a light behind the film. If a diffraction image isobserved, then the analyte is present in the liquid.

In another embodiment of the present invention, a multiple analyte testis constructed on the same support. As shown in FIG. 1, a strip 10 isprovided with several micro-contact printed films 20, 25, 30 and 35,each film having a pattern 40 printed thereon. Each of the micro-contactprinted films 15, 20, 25, and 30 have a different receptor material thatis specific for different analytes. It can be seen that the presentinvention can be formatted in any array with a variety of micro-contactprinted films thereby allowing the user of the biosensor device of thepresent invention to detect the presence of multiple analytes in amedium using a single test.

There are many possible supports for the analyte-specific receptorlayers. Simple physisorption can occur on many materials, such aspolystyrene glass, nylon, or others well known to those of ordinaryskill in the art. Preferred embodiments of immobilizing theanalyte-specific receptor layers have involved covalent attachment, suchas that possible between thiol or disulfide-containing compounds andgold. Typically, a gold coating, 5 to 2000 nm thick, is supported on aSi/SiO₂ wafer, glass, or a polymer film. Optionally, titanium can beused to serve as an adhesion promoter between gold and the support. Theanalyte-specific receptor attaches to the gold surface during contactprinting or immersion from a solution. Preferably, the support comprisesa gold coating on a MYLAR® film.

FIG. 2 outlines the procedure used for microcontact printing. Anelastomeric stamp is used to transfer analyte-specific receptor “ink” toa gold surface by contact; if the stamp is patterned, a patternedanalyte-specific receptor layer forms. The stamp is fabricated bycasting polydimethylsiloxane (PDMS) on a master having the inverse ofthe desired pattern. Masters are prepared using standardphotolithographic techniques, or constructed from existing materialshaving microscale surface features.

In a preferred embodiment of a typical experimental procedure, aphotolithographically produced master is placed in a glass or plasticPetri dish, and a 10:1 ratio (w:w) mixture of SYLGARD® siliconeelastomer 184 and SYLGARD® silicone elastomer 184 curing agent (DowCorning Corporation) is poured over it. The elastomer is allowed to sitfor approximately 30 minutes at room temperature and reduced pressure todegas, then cured for at least 4 hours at 60° C., and gently peeled fromthe master. “Inking” of the elastomeric stamp is accomplished byexposing the stamp to a 0.1 to 10 μM aqueous solution ofdisulfide-derivatized antibody typically by placing the stamp face downin the solution for 10 seconds to 10 minutes. The stamp is allowed todry, either under ambient conditions, or typically by exposure to astream of air or nitrogen gas. Following inking, the stamp is applied toa gold surface. Light pressure is used to ensure complete contactbetween the stamp and the surface. After 1 second to 5 minutes, thestamp is then gently peeled from the surface. Following removal of thestamp, the surface is rinsed and dried. Alternatively, furtherderivatization of unstamped areas can be accomplished, either by using asecond stamp or by exposing the entire surface with a different reagent.Subsequently, exposure to a protein-blocking agent, such as BSA orβ-casein, or any other agent well known in the art, can also be done.

The elastomeric character of the stamp is important to the success ofthe process. Polydimethylsiloxane (PDMS), when cured, is sufficientlyelastomeric to allow good conformal contact of the stamp and thesurface, even for surfaces with significant relief; this contact isessential for efficient contact transfer of the receptor to a gold film.The elastomeric properties of PDMS are also important when the stamp isremoved from the master: if the stamp were rigid (as is the master) itwould be difficult to separate the stamp and master after curing withoutdamaging one of the two substrates. PDMS is also sufficiently rigid toretain its shape, even for features with sub-micron dimension. The stampis durable in that the same stamp can be used over 200 times over aperiod of a year without significant degradation in performance. Using aprinting roll for the stamp could allow for a continuous printingoperation. Alternatively, ink-jet printing of the desired pattern couldalso be done if capable of producing the feature sizes needed fordiffraction, for example ≦100 μm.

A more detailed description of the methods and compositions of thepresent invention follows. All publications cited herein areincorporated by reference in their entirety.

Any plastic film is suitable for the present invention. Preferably, theplastic film is also capable of having a metal coating depositedthereon. These include, but are not limited to polymers such as:polyethylene-terephthalate (e.g., MYLAR®),acrylonitrile-butadiene-styrene, acrylonitrile-methyl acrylatecopolymer, cellophane, cellulosic polymers such as ethyl cellulose,cellulose acetate, cellulose acetate butyrate, cellulose propionate,cellulose triacetate, cellulose triacetate, polyethylene,polyethylene-vinyl acetate copolymers, ionomers (ethylene polymers)polyethylene-nylon copolymers, polypropylene, methyl pentene polymers,polyvinyl fluoride, and aromatic polysulfones. Preferably, the plasticfilm has an optical transparency of greater than 80%. Other suitableplastics and suppliers may be found, for example, in reference workssuch as the Modern Plastics Encyclopedia (McGraw-Hill Publishing Co.,New York 1923-1996).

In one embodiment of the invention, the polymer film has a metal coatingthereon and has an optical transparency of between approximately 5% and95%. A more desired optical transparency for the plastic film used inthe present invention is between approximately 20% and 80%. In a desiredembodiment of the present invention, the polymer film has at least anapproximately 80% optical transparency, and the thickness of the metalcoating is such as to maintain an optical transparency greater thanabout 60%, so that diffraction images can be produced by transmittedlight. This corresponds to a metal coating thickness of about 10 nm.However, in other embodiments of the invention, the gold thickness maybe between approximately 1 nm and 1000 nm; for example, thicker goldcoatings (>20 nm) would still be suitable for producing diffractionimages by reflected light.

The preferred metal for deposition on the film is gold. However, silver,aluminum, chromium, copper, iron, zirconium, platinum and nickel, aswell as oxides of these metals, may be used.

In principle, any surface with corrugations of appropriate size could beused as masters. The process of microcontact printing starts with anappropriate relief structure, from which an elastomeric stamp is cast.This ‘master’ template may be generated photolithographically, or byother procedures, such as commercially available diffraction gratings.In one embodiment, the stamp may be made from polydimethylsiloxane.

The stamp may be applied in air, or under a fluid capable of preventingexcess diffusion of the receptor material. For large-scale or continuousprinting processes, it is most desirable to print in air.

In one embodiment of the present invention, the pattern is formed on themetalized plastic polymer with the analyte-specific receptor layer.After the stamping process, the metalized areas on the plastic mayoptionally be blocked, for example, with a protein-repelling agent suchas β-casein.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof, which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention.

EXAMPLES Example 1

Antibody-conjugated polystyrene particles were produced by carbodiimidecoupling with ethyldimethylaminodicarbodiimide (EDAC, bottle #3 ofPolysciences kit, Catalog # 19539). For this example, 0.125 mL of a 10%suspension of 0.5 micron diameter blue carboxylated particles (BangsLaboratories, Fishers, Ind., Cat #D0005070CB) were activated with anaqueous solution of the EDAC for 1-4 hours, rinsed, then exposed to 300micrograms of a monoclonal antibody to luteinizing hormone, alphasubunit, (Fitzgerald Industries, Concord, Mass., Cat# 10-L10, Clone #M94136). The particles were again rinsed, blocked with bovine serumalbumin, and stored at 2.5% concentration in phosphate buffered saline.

Next, a gold/MYLAR® film was pre-treated (or blocked) with a 5 mg/mLbeta casein solution for 10 minutes, then thoroughly rinsed and driedunder an air stream. A PDMS stamp of 10-micron circles was coated withthiolated antibody by placing the stamp face down in a 0.5 mg/mLthiolated antibody solution and soaking for 10 minutes. A strong airstream was used to thoroughly dry the surface of the stamp. The coatedstamp was placed in contact with the gold/MYLAR® film for 5 minutes,then removed. The resulting printed gold/MYLAR® film was rinsed indistilled water, and dried.

A 10 mM stock solution of the sulfosuccinimidyl6-[3′-(2-pyridyldithio)-propionamido]hexanoate (Sulfo-LC-SPDP) isprepared by dissolving 1.3 mg Sulfo-LC-SPDP into 2.07 ml de-ionizedwater. The conjugation reaction is carried out in phosphate bufferedsaline (PBS) containing 20 mM sodium phosphate buffer, 150 mM NaCl, 1 mMEDTA and 0.02% sodium azide at pH 7.5. One milligram of lyophilizedantibody is dissolved in 450 ml PBS, and 50 ml of Sulfo-LC-SPDP stocksolution is added to the antibody solution. The mixture is allowed toreact at room temperature for 60 minutes. The sample is applied to a 5ml desalting polyacrylamide column previously equilibrated with 5 bedvolumes (25 ml) of PBS. Fractions are eluted using PBS as the elutionbuffer, and the protein in the fractions is monitored using a COOMASSIE®Protein Assay (Pierce Chemical Co). Typically, 50 ul of the COOMASSIE®reagent is mixed with 50 ul of each fraction in a micro-titer plate. TheCOOMASSIE® Blue substrate reacts with the protein, producing a bluecolor, the intensity of which is dependent upon the amount of proteinpresent in the fraction. The fractions which produce the most intenseblue color are those containing the majority of the protein eluted.These fractions are pooled together to produce the disulfide form of thefinal derivatized product. This is typically the form used for contactprinting.

Optionally, the disulfide-pyridyl group present on the disulfide form ofthe thiolated binder can be reduced to a thiol group in a reductionreaction. Instead of desalting on a column equilibrated with PBS, thederivatized protein is desalted on a column equilibrated with an acetatebuffer (100 mM sodium acetate buffer, 100 mM NaCl, pH 4.5). The acidicpH of this acetate buffer acts to protect any disulfide bonds present onthe native protein from undesired reduction. In the reduction reaction,12 mg of dithiothreitol (DTT) is dissolved in 500 ml acetate buffer andadded to 1 ml of the SPDP derivatized protein. The reaction mixture isincubated for 30 minutes at room temperature, and desalted on a 5 mldesalting column equilibrated with 5 bed volumes (25 ml) of acetatebuffer. The protein content of the fractions eluted is again monitoredby the COOMASSIE® Protein Assay as described above, and the fractionscontaining the greatest amount of protein are pooled.

Both the disulfide and reduced forms of the thiolated binders are storedin aqueous solution at 4° C. until used for contact printing.

The sensors were then used to detect an analyte. The analyte solutionwas then mixed with microparticles (typically 50-70 microliters ofanalyte solution in 1% bovine serum albumin with 10-25 microliters of1.5-2.5% particle suspension; preferably, there is a 50:25 ratio ofanalyte solution to particle suspension), and placed on top of a 1 cmsquare sensor sample. After 5 minutes, a nitrocellulose disk (5 or 8micron pore size, Sigma #N3771 or N4146) with a small hole (e.g.{fraction (3/16)}″) punched out of the center was placed on top of thesensor. The disk was used to wick away excess fluid and unboundmicroparticles. At this time, a point light source was transmittedthrough the sensor sample (using the small hole in the nitrocellulose).A diffraction image would be seen on the other side of the light beam inthe presence of the target analyte.

As seen in FIG. 4, SEM photomicrographs showed the patterned placementof the microparticles.

Example 2

A PDMS stamp of an x,y array of 10-micron circles was “inked” withthiolated 30-mer oligonucleotide which is complementary to the targetDNA strand (“30-mer”; base sequence of thiolspacer-5′-CAATCCACGTCACGGACAGGGTGAGGAAGA-3′ sequence ID. No. 1 made byGenosys, Inc., The Woodlands, Tex.) by placing the stamp face down withweight in oven-dried (50° C., vacuum) mixture of the 30-mer and ethylacetate on glass. After 10 minutes, the inked stamp was removed. At thesame time, a gold/MYLAR® film was pre-heated on a 60° C. hot plate for 5minutes. Printing was done by placing the inked PDMS stamp on top of thegold-coated side of MYLAR® at 60° C.; weight and heat were maintainedduring the 5 minute contact time. At this point, the stamp was removedand the printed gold/MYLAR® film was washed with distilled water, andair-dried. The gold/MYLAR® film sample was then blocked with a 2.5 mg/mLbeta casein solution (in phosphate buffered saline, pH 7.2) for 10minutes, and rinsed with distilled water and air-dried.

These sensors were used to test for target DNA. Hybridization of thetarget DNA to the capture DNA patterned on the sensor surface took placeas follows: A pre-heated analyte solution (60° C. water bath, 2 minutes)containing a DNA strand of interest (a biotinylated 70-mer from Genosyswith base sequence of biotin-5′-GGTAGACCGGAGAGCTGTGTCACCATGTGGGTCCCGG,TTGTCTTCCTCACCCTGTCCGTGACGTGGATTG-3′) sequence ID. No. 2 was added to apre-heated sensor (60° C. hot plate, 5 minutes) and then 75 microliterswas added to an approximately 1 cm square sensor for an additional 10minutes heating. After this time, the sensor sample was rinsed withwater, and air-dried for subsequent testing with microparticles. Onevariation to this method is that the analyte solution, e.g., during aPCR amplification, and the microparticles are exposed to the sensor atthe same time.

Next, Streptavidin-coated, 1 micron diameter particles from BangsLaboratories (Catalog # CP01N) were added in 20-30 microliter amounts,concentration of 2.4×10¹¹ particles per mL, to the sensor. The sensorand particles were heated on a 60 ° C. hot plate for 10 minutes(covered, while ensuring that complete evaporation did not take place),and then rinsed gently with distilled water. After this, a point lightsource was transmitted through the sensor sample. A diffraction imagewould be seen on the other side of the light beam in the presence of theDNA analyte.

SEM photomicrographs show the patterned placement of the microparticles.

Example 3

Antibody-conjugated polystyrene particles were produced by carbodiimidecoupling with ethyldimethylaminodicarbodiimide (“EDAC”, bottle #3 ofPolysciences kit, Catalog # 19539). For example, 0.125 mL of a 10%suspension of 0.3 micron diameter blue carboxylated particles (BangsLaboratories, Cat #DC02/1836) were activated with an aqueous solution ofthe EDAC for 1-4 hours, rinsed, then exposed to 300 micrograms of apolyclonal antibody to IgE (Fitzgerald Industries, Cat#20-IR77). Theparticles were again rinsed, blocked with bovine serum albumin, andstored at 1.7% concentration in phosphate buffered saline.

Next, a gold/MYLAR® film was pre-treated (or blocked) with a 5 mg/mLbeta casein solution for 10 minutes, then thoroughly rinsed and driedunder an air stream. A PDMS stamp of an x,y array of 10-micron diametercircles was coated with thiolated antibody (antibody was initiallyFitzgerald Catalog #10-I10 then derivatized or “thiolated” usingSulfo-LC-SPDP by Pierce) by placing the stamp face down in a 0.5 mg/mLthiolated antibody solution and soaking for 10 minutes. A strong airstream was used to thoroughly dry the surface of the stamp. The coatedstamp was placed in contact with the gold/MYLAR® film for 5 minutes,then removed. The resulting printed gold/MYLAR® film was rinsed indistilled water, and dried.

The analyte solution was then mixed with microparticles (typically 50-70microliters of analyte solution in 1% bovine serum albumin with 10-25microliters of 1.5-2.5% particle suspension; preferably, there is a50:25 ratio of analyte solution to particle suspension), and placed ontop of a 1 cm square sensor sample. After 5-10 minutes, a nitrocellulosedisk (5 or 8 micron pore size, Sigma #N3771 or N4146) with a small(e.g., {fraction (3/16)}″ diameter) hole punched out of the center isplaced on top of the sensor. The disk was used to wick away excess fluidand unbound microparticles. At this time, a point light source wastransmitted through the sensor sample by taking advantage of the smallhole in the nitrocellulose. A high order diffraction image was seen onthe other side of the light beam, signifying the presence of theanalyte.

Example 4

A gold/MYLAR® film was pre-treated (or blocked) with a 5 mg/mL betacasein solution in phosphate buffered saline (pH˜7.2) for 10 minutes,then thoroughly rinsed and dried under an air stream. A PDMS stamp of10-micron circles was coated with thiolated antibody (e.g., rabbitanti-Candida albicans, Cat # 20-CR04 from Fitzgerald Industries, Inc.)by placing the stamp face down in a 0.5 mg/mL thiolated antibodysolution and soaking for 10 minutes. A strong air stream was used tothoroughly dry the surface of the stamp. The coated stamp was placed incontact with the gold/MYLAR® film for 2 minutes, then removed. Theresulting printed gold/MYLAR® film was rinsed in distilled water, anddried.

The sensor sample was exposed to a 10% dilution in phospate bufferedsaline, pH 7.2 of 40 nm gold particles coated with goat anti-rabbit IgG(gold conjugate was from Polysciences, Catalog # 22705). After one hour,the samples were thoroughly rinsed with distilled water and dried undera nitrogen or air stream. At this point, the samples do not diffract aHeNe laser beam.

The samples were then exposed to silver enhancing reagents from BBI(either BBI International's kit # SEKL 15 (Batch #2575) or large kit #SEKB250 (Batch #2484) were used). A 1:1 v/v ratio of the enhancer andinitiator reagents in the kit were pre-mixed and then immediately placedon top of the gold-particle coated samples. After 10-20 minutes exposure(preferably, 10 minutes), the samples were rinsed with water, dried, andexamined. At this point, the samples diffracted light (either a laserbeam or a point white light source) most likely due to the larger sizeof the silver nucleated around the gold nanoparticles.

Example 5

Samples prepared as per Examples 1 or 4 could also be developed into adiffraction image by exposing it to an enzyme-conjugate secondaryantibody in the presence of the analyte, such that if the analyte ispresent the secondary antibody would bind and cause subsequentprecipitate development with a precipitating substrate specific to theenzyme.

A gold/MYLAR® film was pre-treated (or blocked) with a 5 mg/mL betacasein solution in phosphate buffered saline (pH˜7.2) for 10 minutes,then thoroughly rinsed and dried under an air stream. A PDMS stamp of10-micron circles was coated with thiolated antibody (e.g., mouseanti-luteinizing hormone beta, Cat # 10-L15 from Fitzgerald Industries,Inc.) by placing the stamp face down in a ˜0.3 mg/mL thiolated antibodysolution and soaking for 10 minutes. A strong air stream was used tothoroughly dry the surface of the stamp. The coated stamp was placed incontact with the gold/MYLAR film for 5 minutes, then removed. Theresulting printed gold/MYLAR® film was rinsed in distilled water, anddried.

The sensor sample was exposed to an analyte solution of luteinizinghormone (Cat # 30-AL15 from Fitzgerald Industries, Inc.) in 1% bovineserum albumin, phosphate buffered saline. Concentration of antigen wasvaried from 0.1 to 1000 ng/mL. After one hour at room temperature, thesample was rinsed with 0.02% TWEEN 20 solution, then distilled water. Asubsequent exposure to a secondary antibody (Fitzgerald Catalog # 61-L05diluted 1:100 in distilled water) for one hour was done, followed byrinsing as above. A TMB membrane enhancer solution (e.g., a 10:1 v/vmixture of Kirkegaard and Perry Laboratories' reagents Cat #50-76-18 andCat#50-77-01) was placed on the sample for 10 minutes to causedevelopment of a blue precipitate in the circles or features. Thisprecipitate caused a diffraction image to form upon irradiation with apoint light source.

2 1 30 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 1 caatccacgt cacggacagg gtgaggaaga 30 2 70 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 2 ggtagaccgg agagctgtgt caccatgtgg gtcccggttg tcttcctcaccctgtccgtg 60 acgtggattg 70

We claim:
 1. A method of detecting an analyte in a medium comprising:adding a diffraction enhancing element to the medium suspected ofcontaining the analyte, wherein the diffraction enhancing element has areceptor material thereon that is specific for the analyte; contactingthe medium with a sensing device, the sensing device comprising: apolymer film; and an analyte-specific receptor layer printed in apattern onto the polymer film wherein the analyte-specific receptorlayer has a receptor material thereon that is specific for the analyte,and wherein the receptor layer is not a self-assembling monolayer;transmitting a light through the polymer film; and detecting presence ofthe analyte by detecting a pattern formed by diffraction of thetransmitted light.
 2. The method of claim 1, wherein theanalyte-specific receptor layer is printed in a pattern such that whenthe sensing device binds an analyte, the sensing device diffractstransmitted light to form a diffraction pattern.
 3. The method of claim1, wherein the diffraction pattern is visible to an unaided eye.
 4. Themethod of claim 1, further comprising a metal coating on the polymerfilm and wherein the analyte-specific receptor layer is printed onto themetal coating.
 5. The method of claim 4, wherein the metal is selectedfrom gold, silver, chromium, nickel, platinum, aluminum, iron, copper,gold oxide, chromium oxide or zirconium.
 6. The method of claim 5,wherein the metal is gold.
 7. The method of claim 6, wherein the goldcoating is between approximately 1 nanometer and 1000 nanometers inthickness.
 8. The method of claim 1, wherein the polymer film isselected from polyethylene-terephthalate,acrylonitrile-butadiene-styrene, acrylonitrile-methyl acrylatecopolymer, cellophane, cellulosic polymers such as ethyl cellulose,cellulose acetate, cellulose acetate butyrate, cellulose propionate,cellulose triacetate, polyethylene, polyethylene-vinyl acetatecopolymers, ionomers (ethylene polymers) polyethylene-nylon copolymers,polypropylene, methyl pentene polymers, polyvinyl fluoride, or aromaticpolysulfones.
 9. The method of claim 8, wherein the polymer film ispolyethylene-terephthalate.
 10. The method of claim 1, wherein thepolymer film is optically transparent.
 11. The method of claim 10,wherein the polymer film has an optical transparency between 5% and 95%.12. The method of claim 10, wherein the polymer film has an opticaltransparency between approximately 20% and 80%.
 13. The method of claim1, wherein there are two or more analyte-specific receptor layers witheach layer having different chemical properties.
 14. The method of claim1, wherein the analyte is selected from bacteria, yeast, fungus, virus,rheumatoid factor, IgG, IgM, IgA and IgE antibodies, carcinoembryonicantigen, streptococcus Group A antigen, viral antigens, antigensassociated with autoimmune disease, allergens, tumor antigens,streptococcus Group B antigen, HIV I or HIV II antigen, antibodiesviruses, antigens specific to RSV, an antibody, antigen, enzyme,hormone, polysaccharide, protein, lipid, carbohydrate, drug or nucleicacid, Neisseria meningitides groups A, B, C, Y and W sub 135,Streptococcus pneumoniae, E. coli K1, Haemophilus influenza type B, anantigen derived from microorganisms, a hapten, a drug of abuse, atherapeutic drug, an environmental agent, or antigens specific toHepatitis.
 15. The method of claim 14, wherein the analyte is bacteria,yeast, fungus or virus.
 16. The method of claim 1, wherein the receptormaterial is selected from antigens, antibodies, oligonucleotides,chelators, enzymes, bacteria, yeasts, fungi, viruses, bacterial pili,bacterial flagellar materials, nucleic acids, polysaccharides, lipids,proteins, carbohydrates, metals, hormones or receptors for saidmaterials.
 17. The method of claim 1, wherein the diffraction enhancingelement is selected from glass, cellulose, synthetic polymers orplastics, latex, polystyrene, polycarbonate, bacterial or fungal cells.18. The method of claim 1, wherein the diffraction enhancing element ispolystyrene latex microspheres.
 19. The method of claim 1, furthercomprising the step of applying a blocking material to the non-printedareas of the polymer film.
 20. The method of claim 19, wherein theblocking material is selected from β-casein, an albumin, a surfactant,polyethylene glycol, polyvinyl alcohol, or sulfur derivatives thereof.21. The method of claim 1, wherein the sensing device further comprisesa layer of blocking material on the polymer film through which theanalyte-specific receptor material is printed.
 22. The method of claim21, wherein the blocking material is selected from β-casein, an albumin,a surfactant, polyethylene glycol, polyvinyl alcohol, or sulfurderivatives thereof.
 23. A method of detecting an analyte in a mediumcomprising: adding a diffraction enhancing element to the mediumsuspected of containing the analyte, wherein the diffraction enhancingelement has a receptor material thereon that is specific for theanalyte; contacting the medium with a sensing device, the sensing devicecomprising: a polymer film coated with metal; and an analyte-specificreceptor layer printed in a pattern onto the metal-coated polymer filmwherein the analyte-specific receptor layer has a receptor materialthereon that is specific for the analyte, and wherein the receptor layeris not a self-assembling monolayer; reflecting a light source off asurface of the metal-coated polymer film; and detecting presence of theanalyte by detecting a pattern formed by diffraction of the reflectedlight.
 24. The method claim 23, wherein the analyte-specific receptorlayer is printed in a pattern such that when the sensing device binds ananalyte, the sensing device diffracts reflected light to form adiffraction pattern.
 25. The method of claim 23, wherein the diffractionpattern is visible to an unaided eye.
 26. The method of claim 23,wherein the metal is selected from gold, silver, chromium, nickel,platinum, aluminum, iron, copper, gold oxide, chromium oxide orzirconium.
 27. The method of claim 26, wherein the metal is gold. 28.The method of claim 27, wherein the gold coating is betweenapproximately 1 nanometer and 1000 nanometers in thickness.
 29. Themethod of claim 23, wherein the polymer film is selected frompolyethylene-terephthalate, acrylonitrile-butadiene-styrene,acrylonitrile-methyl acrylate copolymer, cellophane, cellulosic polymerssuch as ethyl cellulose, cellulose acetate, cellulose acetate butyrate,cellulose propionate, cellulose triacetate, polyethylene,polyethylene-vinyl acetate copolymers, ionomers (ethylene polymers)polyethylene-nylon copolymers, polypropylene, methyl pentene polymers,polyvinyl fluoride, or aromatic polysulfones.
 30. The method of claim29, wherein the polymer film is polyethylene-terephthalate.
 31. Themethod of claim 23, wherein there are two or more analyte-specificreceptor layers with each layer having different chemical properties.32. The method of claim 23, wherein the analyte is selected frombacteria, yeast, fungus, virus, rheumatoid factor, IgG, IgM, IgA and IgEantibodies, carcinoembryonic antigen, streptococcus Group A antigen,viral antigens, antigens associated with autoimmune disease, allergens,tumor antigens, streptococcus Group B antigen, HIV I or HIV II antigen,antibodies viruses, antigens specific to RSV, an antibody, antigen,enzyme, hormone, polysaccharide, protein, lipid, carbohydrate, drug ornucleic acid, Neisseria meningitides groups A, B, C, Y and W sub 135,Streptococcus pneumoniae, E. coli K1, Haemophilus influenza type B, anantigen derived from microorganisms, a hapten, a drug of abuse, atherapeutic drug, an environmental agent, or antigens specific toHepatitis.
 33. The method of claim 32, wherein the analyte is bacteria,yeast, fungus or virus.
 34. The method of claim 23, wherein the receptormaterial is selected from antigens, antibodies, oligonucleotides,chelators, enzymes, bacteria, yeasts, fungi, viruses, bacterial pili,bacterial flagellar materials, nucleic acids, polysaccharides, lipids,proteins, carbohydrates, metals, hormones or receptors for saidmaterials.
 35. The method of claim 23, wherein the diffraction enhancingelement is selected from glass, cellulose, synthetic polymers orplastics, latex, polystyrene, polycarbonate, bacterial or fungal cells.36. The method of claim 23, wherein the diffraction enhancing element ispolystyrene latex microspheres.
 37. The method of claim 23, furthercomprising the step of applying a blocking material to the non-printedareas of the metal-coated polymer film.
 38. The method of claim 37,wherein the blocking material is selected from β-casein, an albumin, asurfactant, polyethylene glycol, polyvinyl alcohol, or sulfurderivatives thereof.
 39. The method of claim 23, wherein the sensingdevice further comprises a layer of blocking material on themetal-coated polymer film through which the analyte-specific receptormaterial is printed.
 40. The method of claim 39, wherein the blockingmaterial is selected from β-casein, an albumin, a surfactant,polyethylene glycol, polyvinyl alcohol, or sulfur derivatives thereof.41. A method of detecting an analyte in a medium comprising: adding adiffraction enhancing element to the medium suspected of containing theanalyte, wherein the diffraction enhancing element has a receptormaterial thereon that is specific for the analyte; contacting the mediumwith a sensing device, the sensing device comprising: a polymer filmcoated with metal; and an analyte-specific receptor layer printed in apattern onto the metal-coated polymer film wherein the analyte-specificreceptor layer has a receptor material thereon that is specific for theanalyte, and wherein the receptor layer is not a self-assemblingmonolayer; transmitting a light through the polymer film; and detectingpresence of the analyte by detecting a pattern formed by diffraction ofthe transmitted light.